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1.
Nat Commun ; 12(1): 2283, 2021 04 16.
Artigo em Inglês | MEDLINE | ID: mdl-33863907

RESUMO

Narcolepsy type 1 (NT1) is a chronic neurological disorder having a strong association with HLA-DQB1*0602, thereby suggesting an immunological origin. Increased risk of NT1 has been reported among children or adolescents vaccinated with AS03 adjuvant-supplemented pandemic H1N1 influenza A vaccine, Pandemrix. Here we show that pediatric Pandemrix-associated NT1 patients have enhanced T-cell immunity against the viral epitopes, neuraminidase 175-189 (NA175-189) and nucleoprotein 214-228 (NP214-228), but also respond to a NA175-189-mimic, brain self-epitope, protein-O-mannosyltransferase 1 (POMT1675-689). A pathogenic role of influenza virus-specific T-cells and T-cell cross-reactivity in NT1 are supported by the up-regulation of IFN-γ, perforin 1 and granzyme B, and by the converging selection of T-cell receptor TRAV10/TRAJ17 and TRAV10/TRAJ24 clonotypes, in response to stimulation either with peptide NA175-189 or POMT1675-689. Moreover, anti-POMT1 serum autoantibodies are increased in Pandemrix-vaccinated children or adolescents. These results thus identify POMT1 as a potential autoantigen recognized by T- and B-cells in NT1.


Assuntos
Vírus da Influenza A Subtipo H1N1/imunologia , Vacinas contra Influenza/efeitos adversos , Influenza Humana/prevenção & controle , Manosiltransferases/imunologia , Narcolepsia/imunologia , Adolescente , Animais , Autoanticorpos/sangue , Autoanticorpos/imunologia , Autoantígenos/imunologia , Linfócitos B/imunologia , Antígenos CD4/genética , Estudos de Casos e Controles , Criança , Pré-Escolar , Reações Cruzadas/imunologia , Modelos Animais de Doenças , Epitopos de Linfócito T/imunologia , Feminino , Cadeias beta de HLA-DQ/imunologia , Humanos , Lactente , Vacinas contra Influenza/imunologia , Influenza Humana/imunologia , Influenza Humana/virologia , Masculino , Camundongos Transgênicos , Narcolepsia/sangue , Narcolepsia/induzido quimicamente , Neuraminidase/imunologia , Linfócitos T/imunologia , Proteínas Virais/imunologia , Adulto Jovem
2.
PLoS Pathog ; 16(6): e1008411, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32598381

RESUMO

In the 2014-2015 influenza season a novel neuraminidase (NA) genotype was detected in global human influenza A surveillance. This novel genotype encoded an N-linked glycosylation site at position 245-247 in the NA protein from clade 3c.2a H3N2 viruses. In the years following the 2014-2015 season, this novel NA glycosylation genotype quickly dominated the human H3N2 population of viruses. To assess the effect this novel N-linked glycan has on virus fitness and antibody binding, recombinant viruses with (NA Gly+) or without (NA Gly-) the 245 NA glycan were created. Viruses with the 245 NA Gly+ genotype grew to a significantly lower infectious virus titer on primary, differentiated human nasal epithelial cells (hNEC) compared to viruses with the 245 NA Gly- genotype, but growth was similar on immortalized cells. The 245 NA Gly+ blocked human and rabbit monoclonal antibodies that target the enzymatic site from binding to their epitope. Additionally, viruses with the 245 NA Gly+ genotype had significantly lower enzymatic activity compared to viruses with the 245 NA Gly- genotype. Human monoclonal antibodies that target residues near the 245 NA glycan were less effective at inhibiting NA enzymatic activity and virus replication of viruses encoding an NA Gly+ protein compared to ones encoding NA Gly- protein. Additionally, a recombinant H6N2 virus with the 245 NA Gly+ protein was more resistant to enzymatic inhibition from convalescent serum from H3N2-infected humans compared to viruses with the 245 NA Gly- genotype. Finally, the 245 NA Gly+ protected from NA antibody mediated virus neutralization. These results suggest that while the 245 NA Gly+ decreases virus replication in hNECs and decreases enzymatic activity, the 245 NA glycan blocks the binding of monoclonal and human serum NA specific antibodies that would otherwise inhibit enzymatic activity and virus replication.


Assuntos
Anticorpos Monoclonais/imunologia , Anticorpos Antivirais/imunologia , Vírus da Influenza A Subtipo H3N2/fisiologia , Influenza Humana , Neuraminidase , Proteínas Virais , Replicação Viral , Animais , Cães , Células HEK293 , Humanos , Influenza Humana/genética , Influenza Humana/imunologia , Células Madin Darby de Rim Canino , Neuraminidase/genética , Neuraminidase/imunologia , Coelhos , Proteínas Virais/genética , Proteínas Virais/imunologia , Replicação Viral/genética , Replicação Viral/imunologia
3.
PLoS One ; 15(5): e0233001, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32401814

RESUMO

Antibodies against influenza virus neuraminidase (NA) protein prevent releasing of the virus from host cells and spreading of infection foci and are considered the 'second line of defence' against influenza. Haemagglutinin inhibition antibody-low responders (HI-LRs) are present among influenza split vaccine recipients. The NA inhibition (NAI) antibody response in vaccinees is worth exploring, especially those in the HI-LRs population. We collected pre- and post-vaccination sera from 61 recipients of an inactivated, monovalent, split vaccine against A/H1N1pdm09 and acute and convalescent sera from 49 unvaccinated patients naturally infected with the A/H1N1pdm09 virus during the 2009 influenza pandemic. All samples were subjected to haemagglutinin inhibition (HI), NAI and neutralisation assays. Most paired sera from naturally infected patients exhibited marked elevation in the NAI activity, and seroconversion rates (SCR) among HI-LRs and HI-responders (HI-Rs) were 60% and 87%, respectively; however, those from vaccinees displayed low increase in the NAI activity, and the SCR among HI-LRs and HI-Rs were 0% and 12%, respectively. In both HI-LRs and HI-Rs, vaccination with the inactivated, monovalent, split vaccine failed to elicit the NAI activity efficiently in the sera of the naive population, compared with the natural infection. Hence, the improvement of influenza vaccines is warranted to elicit not only HI but also NAI antibodies.


Assuntos
Anticorpos Antivirais/sangue , Vírus da Influenza A Subtipo H1N1/imunologia , Vacinas contra Influenza/imunologia , Influenza Humana/imunologia , Influenza Humana/prevenção & controle , Neuraminidase/antagonistas & inibidores , Neuraminidase/imunologia , Proteínas Virais/antagonistas & inibidores , Proteínas Virais/imunologia , Adolescente , Adulto , Anticorpos Neutralizantes/sangue , Criança , Pré-Escolar , Feminino , História do Século XXI , Humanos , Influenza Humana/epidemiologia , Japão , Masculino , Pessoa de Meia-Idade , Pandemias/história , Vacinas de Produtos Inativados/imunologia , Adulto Jovem
4.
Arch Virol ; 165(4): 891-911, 2020 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-32060794

RESUMO

Pandemics caused by influenza A virus (IAV) are responsible for the deaths of millions of humans around the world. One of these pandemics occurred in Mexico in 2009. Despite the impact of IAV on human health, there is no effective vaccine. Gene mutations and translocation of genome segments of different IAV subtypes infecting a single host cell make the development of a universal vaccine difficult. The design of immunogenic peptides using bioinformatics tools could be an interesting strategy to increase the success of vaccines. In this work, we used the predicted amino acid sequences of the neuraminidase (NA) and hemagglutinin (HA) proteins of different IAV subtypes to perform multiple alignments, epitope predictions, molecular dynamics simulations, and experimental validation. Peptide selection was based on the following criteria: promiscuity, protein surface exposure, and the degree of conservation among different medically relevant IAV strains. These peptides were tested using immunological assays to test their ability to induce production of antibodies against IAV. We immunized rabbits and mice and measured the levels of IgG and IgA antibodies in serum samples and nasal washes. Rabbit antibodies against the peptides P11 and P14 (both of which are hybrids of NA and HA) recognized HA from both group 1 (H1, H2, and H5) and group 2 (H3 and H7) IAV and also recognized the purified NA protein from the viral stock (influenza A Puerto Rico/916/34). IgG antibodies from rabbits immunized with P11 and P14 were capable of recognizing viral particles and inhibited virus hemagglutination. Additionally, intranasal immunization of mice with P11 and P14 induced specific IgG and IgA antibodies in serum and nasal mucosa, respectively. Interestingly, the IgG antibodies were found to have neutralizing capability. In conclusion, the peptides designed through in silico studies were validated in experimental assays.


Assuntos
Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , Vírus da Influenza A/imunologia , Vacinas contra Influenza/imunologia , Influenza Humana/prevenção & controle , Sequência de Aminoácidos , Animais , Biologia Computacional , Epitopos/química , Epitopos/genética , Epitopos/imunologia , Glicoproteínas de Hemaglutininação de Vírus da Influenza/química , Glicoproteínas de Hemaglutininação de Vírus da Influenza/genética , Glicoproteínas de Hemaglutininação de Vírus da Influenza/imunologia , Humanos , Imunização , Vírus da Influenza A/química , Vírus da Influenza A/genética , Vacinas contra Influenza/química , Vacinas contra Influenza/genética , Influenza Humana/imunologia , Influenza Humana/virologia , Camundongos , Camundongos Endogâmicos BALB C , Neuraminidase/química , Neuraminidase/genética , Neuraminidase/imunologia , Coelhos , Alinhamento de Sequência , Proteínas Virais/química , Proteínas Virais/genética , Proteínas Virais/imunologia
5.
Biochim Biophys Acta Mol Basis Dis ; 1866(5): 165692, 2020 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-31972227

RESUMO

Many important pathogen-host interactions rely on highly specific carbohydrate binding events. In the case of the protozoan Trypanosoma cruzi, the causative agent of Chagas disease, glycointeractions involving sialic acid (SA) residues are pivotal for parasite infectivity, escape from immune surveillance and pathogenesis. Though unable to synthesize SA de novo, T. cruzi displays a unique trans-Sialidase (TS) enzyme, which is able to cleave terminal SA residues from host donor glycoconjugates and transfer them onto parasite surface mucins, thus generating protective/adhesive structures. In addition, this parasite sheds TS into the bloodstream, as a way of modifying the surface SA signature, and thereby the signaling/functional properties of mammalian host target cells on its own advantage. Here, we discuss the pathogenic aspects of T. cruzi TS: its molecular adaptations, the multiplicity of interactions in which it is involved during infections, and the array of novel and appealing targets for intervention in Chagas disease provided by TS-remodeled sialoglycophenotypes.


Assuntos
Doença de Chagas/imunologia , Glicoproteínas/metabolismo , Interações Hospedeiro-Parasita/imunologia , Neuraminidase/metabolismo , Polissacarídeos/imunologia , Proteínas de Protozoários/metabolismo , Trypanosoma cruzi/patogenicidade , Animais , Doença de Chagas/parasitologia , Glicoproteínas/imunologia , Humanos , Evasão da Resposta Imune , Neuraminidase/imunologia , Polissacarídeos/química , Polissacarídeos/metabolismo , Proteínas de Protozoários/imunologia , Ácidos Siálicos/química , Ácidos Siálicos/imunologia , Ácidos Siálicos/metabolismo , Trypanosoma cruzi/enzimologia , Trypanosoma cruzi/imunologia , Fatores de Virulência/imunologia , Fatores de Virulência/metabolismo
6.
J Virol ; 94(7)2020 03 17.
Artigo em Inglês | MEDLINE | ID: mdl-31941786

RESUMO

Despite evidence that antibodies targeting the influenza virus neuraminidase (NA) protein can be protective and are broadly cross-reactive, the immune response to NA during infection is poorly understood compared to the response to hemagglutinin (HA) protein. As such, we compared the antibody profile to HA and NA in two naturally infected human cohorts in Auckland, New Zealand: (i) a serosurvey cohort, consisting of pre- and post-influenza season sera from PCR-confirmed influenza cases (n = 50), and (ii) an immunology cohort, consisting of paired sera collected after PCR-confirmation of infection (n = 94). The induction of both HA and NA antibodies in these cohorts was influenced by age and subtype. Seroconversion to HA was more frequent in those <20 years old (yo) for influenza A (serosurvey, P = 0.01; immunology, P = 0.02) but not influenza B virus infection. Seroconversion to NA was not influenced by age or virus type. Adults ≥20 yo infected with influenza A viruses were more likely to show NA-only seroconversion compared to children (56% versus 14% [5 to 19 yo] and 0% [0 to 4 yo], respectively). Conversely, children infected with influenza B viruses were more likely than adults to show NA-only seroconversion (88% [0 to 4 yo] and 75% [5 to 19 yo] versus 40% [≥20 yo]). These data indicate a potential role for immunological memory in the dynamics of HA and NA antibody responses. A better mechanistic understanding of this phenomenon will be critical for any future vaccines aimed at eliciting NA immunity.IMPORTANCE Data on the immunologic responses to neuraminidase (NA) is lacking compared to what is available on hemagglutinin (HA) responses, despite growing evidence that NA immunity can be protective and broadly cross-reactive. Understanding these NA responses during natural infection is key to exploiting these properties for improving influenza vaccines. Using two community-acquired influenza cohorts, we showed that the induction of both HA and NA antibodies after infection is influenced by age and subtypes. Such response dynamics suggest the influence of immunological memory, and understanding how this process is regulated will be critical to any vaccine effort targeting NA immunity.


Assuntos
Anticorpos Antivirais/sangue , Glicoproteínas de Hemaglutininação de Vírus da Influenza/imunologia , Vírus da Influenza A/imunologia , Vírus da Influenza B/imunologia , Influenza Humana/imunologia , Neuraminidase/imunologia , Adolescente , Adulto , Fatores Etários , Criança , Pré-Escolar , Feminino , Humanos , Memória Imunológica , Lactente , Recém-Nascido , Influenza Humana/sangue , Influenza Humana/epidemiologia , Masculino , Nova Zelândia/epidemiologia , Reação em Cadeia da Polimerase , Estudos Soroepidemiológicos , Adulto Jovem
7.
Influenza Other Respir Viruses ; 14(2): 237-243, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-31837101

RESUMO

BACKGROUND: This report summarizes the discussions and conclusions from the "Immunological Assays and Correlates of Protection for Next-Generation Influenza Vaccines" meeting which took place in Siena, Italy, from March 31, 2019, to April 2, 2019. CONCLUSIONS: Furthermore, we review current correlates of protection against influenza virus infection and disease and their usefulness for the development of next generation broadly protective and universal influenza virus vaccines.


Assuntos
Vacinas contra Influenza/imunologia , Influenza Humana/imunologia , Animais , Anticorpos Neutralizantes/sangue , Anticorpos Antivirais/sangue , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/imunologia , Testes de Inibição da Hemaglutinação , Glicoproteínas de Hemaglutininação de Vírus da Influenza/imunologia , Humanos , Imunidade nas Mucosas , Vírus da Influenza A Subtipo H1N1/imunologia , Vacinas contra Influenza/biossíntese , Influenza Humana/prevenção & controle , Modelos Animais , Neuraminidase/sangue , Neuraminidase/imunologia , Infecções por Orthomyxoviridae/imunologia , Infecções por Orthomyxoviridae/prevenção & controle , Testes Sorológicos/métodos , Vacinação
8.
Proc Natl Acad Sci U S A ; 117(1): 619-628, 2020 01 07.
Artigo em Inglês | MEDLINE | ID: mdl-31843889

RESUMO

Influenza B viruses have circulated in humans for over 80 y, causing a significant disease burden. Two antigenically distinct lineages ("B/Victoria/2/87-like" and "B/Yamagata/16/88-like," termed Victoria and Yamagata) emerged in the 1970s and have cocirculated since 2001. Since 2015 both lineages have shown unusually high levels of epidemic activity, the reasons for which are unclear. By analyzing over 12,000 influenza B virus genomes, we describe the processes enabling the long-term success and recent resurgence of epidemics due to influenza B virus. We show that following prolonged diversification, both lineages underwent selective sweeps across the genome and have subsequently taken alternate evolutionary trajectories to exhibit epidemic dominance, with no reassortment between lineages. Hemagglutinin deletion variants emerged concomitantly in multiple Victoria virus clades and persisted through epistatic mutations and interclade reassortment-a phenomenon previously only observed in the 1970s when Victoria and Yamagata lineages emerged. For Yamagata viruses, antigenic drift of neuraminidase was a major driver of epidemic activity, indicating that neuraminidase-based vaccines and cross-reactivity assays should be employed to monitor and develop robust protection against influenza B morbidity and mortality. Overall, we show that long-term diversification and infrequent selective sweeps, coupled with the reemergence of hemagglutinin deletion variants and antigenic drift of neuraminidase, are factors that contributed to successful circulation of diverse influenza B clades. Further divergence of hemagglutinin variants with poor cross-reactivity could potentially lead to circulation of 3 or more distinct influenza B viruses, further complicating influenza vaccine formulation and highlighting the urgent need for universal influenza vaccines.


Assuntos
Doenças Transmissíveis Emergentes/virologia , Epidemias/prevenção & controle , Evolução Molecular , Vírus da Influenza B/genética , Vacinas contra Influenza/uso terapêutico , Influenza Humana/virologia , Doenças Transmissíveis Emergentes/epidemiologia , Doenças Transmissíveis Emergentes/imunologia , Doenças Transmissíveis Emergentes/prevenção & controle , Variação Genética , Genoma Viral/genética , Glicoproteínas de Hemaglutininação de Vírus da Influenza/genética , Humanos , Vírus da Influenza B/imunologia , Vírus da Influenza B/patogenicidade , Influenza Humana/epidemiologia , Influenza Humana/imunologia , Influenza Humana/prevenção & controle , Neuraminidase/genética , Neuraminidase/imunologia , Seleção Genética/imunologia
9.
J Virol ; 94(4)2020 01 31.
Artigo em Inglês | MEDLINE | ID: mdl-31748388

RESUMO

The majority of antibodies induced by influenza neuraminidase (NA), like those against hemagglutinin (HA), are relatively specific to viruses isolated within a limited time window, as seen in serological studies and the analysis of many murine monoclonal antibodies (MAbs). We report three broadly reactive human MAbs targeting N1 NA. Two were isolated from a young adult vaccinated with trivalent influenza vaccine (TIV), which inhibited N1 NA from viruses isolated from humans over a period of a hundred years. The third antibody, isolated from a child with acute mild H7N9 infection, inhibited both group 1 N1 and group 2 N9 NAs. In addition, the antibodies cross-inhibited the N1 NAs of highly pathogenic avian H5N1 influenza viruses. These antibodies are protective in prophylaxis against seasonal H1N1 viruses in mice. This study demonstrates that human antibodies to N1 NA with exceptional cross-reactivity can be recalled by vaccination and highlights the importance of standardizing the NA antigen in seasonal vaccines to offer optimal protection.IMPORTANCE Antibodies to the influenza virus NA can provide protection against influenza disease. Analysis of human antibodies to NA lags behind that of antibodies to HA. We show that human monoclonal antibodies against NA induced by vaccination and infection can be very broadly reactive, with the ability to inhibit a wide spectrum of N1 NAs on viruses isolated between 1918 and 2018. This suggests that antibodies to NA may be a useful therapy and that the efficacy of influenza vaccines could be enhanced by ensuring the appropriate content of NA antigen.


Assuntos
Proteção Cruzada/imunologia , Vacinas contra Influenza/imunologia , Influenza Humana/imunologia , Neuraminidase/imunologia , Animais , Anticorpos Monoclonais/imunologia , Criança , Reações Cruzadas/imunologia , Cães , Feminino , Células HEK293 , Hemaglutininas/imunologia , Humanos , Imunização Passiva , Vírus da Influenza A Subtipo H1N1/imunologia , Virus da Influenza A Subtipo H5N1/imunologia , Subtipo H7N9 do Vírus da Influenza A/imunologia , Células Madin Darby de Rim Canino , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos DBA , Neuraminidase/metabolismo , Infecções por Orthomyxoviridae/virologia , Vacinação , Adulto Jovem
10.
Viruses ; 11(11)2019 11 10.
Artigo em Inglês | MEDLINE | ID: mdl-31717638

RESUMO

H5Nx viruses have continuously emerged in the world, causing poultry industry losses and posing a potential public health risk. Here, we studied the phylogeny, pathogenicity, transmission, and immune response of four H5N6 avian influenza viruses in chickens and mice, which were isolated from waterfowl between 2013 and 2014. Their HA genes belong to Clade 2.3.4.4, circulated in China since 2008. Their NA genes fall into N6-like/Eurasian sublineage. Their internal genes originated from different H5N1 viruses. The results suggested that the four H5N6 viruses were reassortants of the H5N1 and H6N6 viruses. They cause lethal infection with high transmission capability in chickens. They also cause mild to severe pathogenicity in mice and can spread to the brain through the blood-brain barrier. During the infection, the viruses result in the up-regulation of PRRs and cytokine in brains and lungs of chickens and mice. Our results suggested that the high viral loads of several organs may result in disease severity in chickens and mice; there were varying levels of cytokines induced by the H5N6 viruses with different pathogenicity in chickens and mice.


Assuntos
Interações Hospedeiro-Patógeno/imunologia , Vírus da Influenza A/classificação , Vírus da Influenza A/fisiologia , Infecções por Orthomyxoviridae/imunologia , Infecções por Orthomyxoviridae/transmissão , Infecções por Orthomyxoviridae/virologia , Filogenia , Animais , Galinhas , Citocinas/metabolismo , Genes Virais , Glicoproteínas de Hemaglutininação de Vírus da Influenza/genética , Glicoproteínas de Hemaglutininação de Vírus da Influenza/imunologia , Camundongos , Neuraminidase/genética , Neuraminidase/imunologia , Infecções por Orthomyxoviridae/metabolismo , Doenças das Aves Domésticas/imunologia , Doenças das Aves Domésticas/transmissão , Doenças das Aves Domésticas/virologia , Receptores de Reconhecimento de Padrão/metabolismo , Proteínas Virais/genética , Proteínas Virais/imunologia , Eliminação de Partículas Virais
11.
Cell Host Microbe ; 26(6): 715-728.e8, 2019 12 11.
Artigo em Inglês | MEDLINE | ID: mdl-31757769

RESUMO

H7N9 avian influenza virus causes severe infections and might have the potential to trigger a major pandemic. Molecular determinants of human humoral immune response to N9 neuraminidase (NA) proteins, which exhibit unusual features compared with seasonal influenza virus NA proteins, are ill-defined. We isolated 35 human monoclonal antibodies (mAbs) from two H7N9 survivors and two vaccinees. These mAbs react to NA in a subtype-specific manner and recognize diverse antigenic sites on the surface of N9 NA, including epitopes overlapping with, or distinct from, the enzyme active site. Despite recognizing multiple antigenic sites, the mAbs use a common mechanism of action by blocking egress of nascent virions from infected cells, thereby providing an antiviral prophylactic and therapeutic protection in vivo in mice. Studies of breadth, potency, and diversity of antigenic recognition from four subjects suggest that vaccination with inactivated adjuvanted vaccine induce NA-reactive responses comparable to that of H7N9 natural infection.


Assuntos
Anticorpos Neutralizantes , Subtipo H7N9 do Vírus da Influenza A/imunologia , Neuraminidase/imunologia , Infecções por Orthomyxoviridae , Liberação de Vírus/efeitos dos fármacos , Animais , Anticorpos Heterófilos/farmacologia , Anticorpos Monoclonais/farmacologia , Anticorpos Neutralizantes/farmacologia , Anticorpos Antivirais/farmacologia , Aves , Epitopos/imunologia , Humanos , Subtipo H7N9 do Vírus da Influenza A/efeitos dos fármacos , Vacinas contra Influenza/imunologia , Influenza Aviária/prevenção & controle , Influenza Aviária/virologia , Influenza Humana/prevenção & controle , Influenza Humana/virologia , Camundongos , Infecções por Orthomyxoviridae/prevenção & controle , Infecções por Orthomyxoviridae/virologia , Profilaxia Pré-Exposição , Vacinação , Vacinas de Produtos Inativados , Proteínas Virais/imunologia
12.
PLoS One ; 14(10): e0223784, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31609986

RESUMO

Currently, influenza vaccine manufacturers need to produce 1-5 x 107 PFU of each vaccine strain to fill one dose of the current live-attenuated-influenza-vaccine (LAIV). To make a single dose of inactivated vaccine (15 ug of each hemagglutinin), the equivalent of 1010 PFU of each vaccine strains need to be grown. This high dose requirement is a major drawback for manufacturing as well as rapidly sourcing sufficient doses during a pandemic. Using our computer-aided vaccine platform Synthetic Attenuated Virus Engineering (SAVE), we created a vaccine candidate against pandemic H1N1 A/CA/07/2009 (CodaVax-H1N1) with robust efficacy in mice and ferrets, and is protective at a much lower dose than the current LAIV. CodaVax-H1N1 is currently in Phase I/II clinical trials. The hemagglutinin (HA) and neuraminidase (NA) gene segments of A/California/07/2009 (H1N1) (CA07) were "de-optimized" and a LAIV was generated ex silico using DNA synthesis. In DBA/2 mice, vaccination at a very low dose (100 or approximately 1 PFU) with CodaVax-H1N1 prevented disease after lethal challenge with wild-type H1N1. In BALB/c mice, as little as 103 PFU was protective against lethal challenge with mouse-adapted H1N1. In ferrets, CodaVax-H1N1 was more potent compared to currently licensed LAIV and still effective at a low dose of 103 PFU at preventing replication of challenge virus.


Assuntos
Glicoproteínas de Hemaglutininação de Vírus da Influenza/genética , Vírus da Influenza A Subtipo H1N1/imunologia , Vacinas contra Influenza/administração & dosagem , Neuraminidase/genética , Proteínas Virais/genética , Animais , Desenho Assistido por Computador , Modelos Animais de Doenças , Furões , Glicoproteínas de Hemaglutininação de Vírus da Influenza/imunologia , Vírus da Influenza A Subtipo H1N1/genética , Vacinas contra Influenza/genética , Vacinas contra Influenza/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos DBA , Neuraminidase/imunologia , Vacinação , Vacinas Atenuadas/administração & dosagem , Vacinas Atenuadas/imunologia , Proteínas Virais/imunologia
13.
mBio ; 10(5)2019 10 22.
Artigo em Inglês | MEDLINE | ID: mdl-31641082

RESUMO

Influenza virus neuraminidase (NA) has been under intense study recently as a vaccine antigen, yet there remain unanswered questions regarding the immune response directed toward NA. Antibodies (Abs) that can inhibit NA activity have been shown to aid in the control of disease caused by influenza virus infection in humans and animal models, yet how and if interactions between the Fc portion of anti-NA Abs and Fcγ receptors (FcγR) contribute to protection has not yet been extensively studied. Herein, we show that poly- and monoclonal anti-NA IgG antibodies with NA inhibitory activity can control A(H1N1)pdm09 infection in the absence of FcγRs, but FcγR interaction aided in viral clearance from the lungs. In contrast, a mouse-human chimeric anti-NA IgG1 that was incapable of mediating NA inhibition (NI) solely relied on FcγR interaction to protect transgenic mice (with a humanized FcγR compartment) against A(H1N1)pdm09 infection. As such, this study suggests that NA-specific antibodies contribute to protection against influenza A virus infection even in the absence of NI activity and supports protection through multiple effector mechanisms.IMPORTANCE There is a pressing need for next-generation influenza vaccine strategies that are better able to manage antigenic drift and the cocirculation of multiple drift variants and that consistently improve vaccine effectiveness. Influenza virus NA is a key target antigen as a component of a next-generation vaccine in the influenza field, with evidence for a role in protective immunity in humans. However, mechanisms of protection provided by antibodies directed to NA remain largely unexplored. Herein, we show that antibody Fc interaction with Fcγ receptors (FcγRs) expressed on effector cells contributes to viral control in a murine model of influenza. Importantly, a chimeric mouse-human IgG1 with no direct antiviral activity was demonstrated to solely rely on FcγRs to protect mice from disease. Therefore, antibodies without NA enzymatic inhibitory activity may also play a role in controlling influenza viruses and should be of consideration when designing NA-based vaccines and assessing immunogenicity.


Assuntos
Anticorpos Antivirais/farmacologia , Anticorpos Antivirais/uso terapêutico , Antivirais/farmacologia , Antivirais/uso terapêutico , Vírus da Influenza A Subtipo H1N1/efeitos dos fármacos , Neuraminidase/imunologia , Infecções por Orthomyxoviridae/tratamento farmacológico , Animais , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/farmacologia , Anticorpos Monoclonais/uso terapêutico , Anticorpos Antivirais/imunologia , Antivirais/imunologia , Feminino , Fragmentos Fc das Imunoglobulinas/imunologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Infecções por Orthomyxoviridae/imunologia
14.
Science ; 366(6464): 499-504, 2019 10 25.
Artigo em Inglês | MEDLINE | ID: mdl-31649200

RESUMO

Better vaccines against influenza virus are urgently needed to provide broader protection against diverse strains, subtypes, and types. Such efforts are assisted by the identification of novel broadly neutralizing epitopes targeted by protective antibodies. Influenza vaccine development has largely focused on the hemagglutinin, but the other major surface antigen, the neuraminidase, has reemerged as a potential target for universal vaccines. We describe three human monoclonal antibodies isolated from an H3N2-infected donor that bind with exceptional breadth to multiple different influenza A and B virus neuraminidases. These antibodies neutralize the virus, mediate effector functions, are broadly protective in vivo, and inhibit neuraminidase activity by directly binding to the active site. Structural and functional characterization of these antibodies will inform the development of neuraminidase-based universal vaccines against influenza virus.


Assuntos
Anticorpos Monoclonais/imunologia , Anticorpos Antivirais/imunologia , Neuraminidase/imunologia , Proteínas Virais/imunologia , Adulto , Animais , Domínio Catalítico , Linhagem Celular , Epitopos/imunologia , Feminino , Humanos , Imunização Passiva , Vírus da Influenza A Subtipo H3N2/imunologia , Vírus da Influenza A/enzimologia , Vírus da Influenza B/enzimologia , Vacinas contra Influenza , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos DBA , Testes de Neutralização , Estrutura Quaternária de Proteína
15.
Front Immunol ; 10: 2360, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31649671

RESUMO

Altered sialylation is generally maintained by a fine balance between sialidases and sialyltransferases, which plays an essential role during disease pathogenesis. TLR4 is a membrane-bound highly sialylated glycoprotein predominantly having α2,3-linked sialic acids. It is one of the most important client molecules in the anti-leishmanial innate immune arm. Here, we initiated a comprehensive study on the modulation of TLR4 sialylation in Leishmania donovani (L. d)-infected macrophages by a mammalian sialidase/neuraminidase-1 (Neu1) having substrate specificity toward α2,3-linked sialic acids. We observed reduced membrane-associated Neu1 with its decreased enzyme activity in infected macrophages. Moreover, we demonstrated reduced association of Neu1 with TLR4 leading to enhanced sialylation of TLR4 in these infected cells. Conversely, Neu1 over expression exhibited enhanced association of TLR4 with Neu1 leading to reduced sialylation which possibly linked to increased association of TLR4 with its downstream adaptor protein, MyD88. This, in turn, activated downstream MAP kinase signaling pathway, with enhanced nuclear translocation of NFκB that resulted in increased genetic and protein levels expression of Th1 cytokines and effector molecule nitric oxide secretion which ultimately leads to reduced parasite burden in macrophages. This was further validated by Neu1 silencing in infected macrophages which reversed such a situation. Such events strongly confirm the importance of Neu1 in modulation of TLR4 sialylation during parasite infection resulting in impairment of innate immune response. Furthermore, decreased membrane-bound Neu1 in infected macrophages could be attributed to its reduced tyrosine-phosphorylation as well as diminished association with cathepsin A. Both these phenomenon possibly play significant roles in inhibiting translocation of the sialidase from cytosol to membrane. Taken together, our study first time demonstrated impaired translocation of cytosolic Neu1 to the membrane of L. donovani-infected macrophages due to impaired phosphorylation of this enzyme. This novel finding establishes a link between enhanced α2,3-linked sialic acids on TLR4 and reduced membrane-bound Neu1 which plays a significant role for inhibiting downstream signaling to establish successful infection in the host cells.


Assuntos
Leishmania donovani/imunologia , Leishmaniose Visceral , Sistema de Sinalização das MAP Quinases/imunologia , Macrófagos , Neuraminidase/imunologia , Receptor 4 Toll-Like/imunologia , Animais , Leishmaniose Visceral/imunologia , Leishmaniose Visceral/patologia , Macrófagos/imunologia , Macrófagos/parasitologia , Macrófagos/patologia , Mesocricetus , Camundongos , Camundongos Endogâmicos BALB C , Ácidos Siálicos/imunologia
16.
Artigo em Inglês | MEDLINE | ID: mdl-31649895

RESUMO

There is an unmet public health need for a universal influenza vaccine (UIV) to provide broad and durable protection from influenza virus infections. The identification of broadly protective antibodies and cross-reactive T cells directed to influenza viral targets present a promising prospect for the development of a UIV. Multiple targets for cross-protection have been identified in the stalk and head of hemagglutinin (HA) to develop a UIV. Recently, neuraminidase (NA) has received significant attention as a critical component for increasing the breadth of protection. The HA stalk-based approaches have shown promising results of broader protection in animal studies, and their feasibility in humans are being evaluated in clinical trials. Mucosal immune responses and cross-reactive T cell immunity across influenza A and B viruses intrinsic to live attenuated influenza vaccine (LAIV) have emerged as essential features to be incorporated into a UIV. Complementing the weakness of the stand-alone approaches, prime-boost vaccination combining HA stalk, and LAIV is under clinical evaluation, with the aim to increase the efficacy and broaden the spectrum of protection. Preexisting immunity in humans established by prior exposure to influenza viruses may affect the hierarchy and magnitude of immune responses elicited by an influenza vaccine, limiting the interpretation of preclinical data based on naive animals, necessitating human challenge studies. A consensus is yet to be achieved on the spectrum of protection, efficacy, target population, and duration of protection to define a "universal" vaccine. This review discusses the recent advancements in the development of UIVs, rationales behind cross-protection and vaccine designs, and challenges faced in obtaining balanced protection potency, a wide spectrum of protection, and safety relevant to UIVs.


Assuntos
Proteção Cruzada/imunologia , Vacinas contra Influenza/imunologia , Influenza Humana/prevenção & controle , Anticorpos Antivirais/imunologia , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/metabolismo , Reações Cruzadas/imunologia , Epitopos de Linfócito T/imunologia , Glicoproteínas de Hemaglutininação de Vírus da Influenza/imunologia , Humanos , Vacinas contra Influenza/normas , Influenza Humana/imunologia , Influenza Humana/virologia , Neuraminidase/química , Neuraminidase/imunologia , Vacinas Atenuadas/imunologia , Vacinas Atenuadas/normas , Vacinologia/métodos , Vacinologia/normas , Proteínas Virais/química , Proteínas Virais/imunologia
17.
Vaccine ; 37(42): 6154-6161, 2019 09 30.
Artigo em Inglês | MEDLINE | ID: mdl-31495597

RESUMO

The clade 2·3·4·4 H5Nx is a highly pathogenic avian influenza (HPAI) virus, which first appeared in China and has spread worldwide since then, including Korea. It is divided into subclades a - d, but the PR8-derived recombinant clade 2·3·4·4 a viruses replicate inefficiently in embryonated chicken eggs (ECEs). High virus titer in ECEs and no mammalian pathogenicity are the most important prerequisites of efficacious and safer vaccine strains against HPAI. In this study, we have synthesized hemagglutinin (HA) and neuraminidase (NA) genes based on the consensus amino acid sequences of the clade 2·3·4·4a and b H5N8 HPAIVs, using the GISAID database. We generated PR8-derived H5N8 recombinant viruses with single point mutations in HA and NA, which are related to efficient replication in ECEs. The H103Y mutation in HA increased mammalian pathogenicity as well as virus titer in ECEs, by 10-fold. We also successfully eradicated mammalian pathogenicity in H103Y-bearing H5N8 recombinant virus by exchanging PB2 genes of PR8 and 01310 (Korean H9N2 vaccine strain). The final optimized H5N8 vaccine strain completely protected against a heterologous clade 2·3·4·4c H5N6 HPAIV in chickens, and induced hemagglutination inhibition (HI) antibody in ducks. However, the antibody titer of ducks showed age-dependent results. Thus, H103Y and 01310PB2 gene have been successfully applied to generate a highly productive, safe, and efficacious clade 2·3·4·4 H5N8 vaccine strain in ECEs.


Assuntos
Glicoproteínas de Hemaglutininação de Vírus da Influenza/imunologia , Vírus da Influenza A Subtipo H5N8/imunologia , Vacinas contra Influenza/imunologia , Influenza Aviária/prevenção & controle , Neuraminidase/imunologia , Vacinas Sintéticas/imunologia , Sequência de Aminoácidos , Animais , Anticorpos Antivirais/sangue , Bioengenharia , Embrião de Galinha , Galinhas/imunologia , Patos/imunologia , Glicoproteínas de Hemaglutininação de Vírus da Influenza/genética , Vacinas contra Influenza/genética , Influenza Aviária/imunologia , Influenza Aviária/virologia , Neuraminidase/genética , Mutação Puntual/genética
18.
ACS Infect Dis ; 5(11): 1813-1819, 2019 11 08.
Artigo em Inglês | MEDLINE | ID: mdl-31538468

RESUMO

trans-Sialidase and cruzipain are important virulence factors from Trypanosoma cruzi, the etiological agent of Chagas disease, that have highly antigenic domains in their structure and were reported as potential tools for diagnosis of the illness. The aim of the present study is to assess the possibility of using cruzipain and the catalytic domain of trans-sialidase in a Surface Plasmon Resonance-based immunosensor for the diagnosis of chronic Chagas disease. Immunoassays carried out with canine sera verified that cruzipain allows the detection of anti-Trypanosoma cruzi antibodies whereas recombinant trans-sialidase did not yield specific detections, due to the high dilutions of serum used in the immunoassays that hinder the possibility to sense the specific low titer antibodies. The developed cruzipain-based biosensor, whose price per assay is comparable to a commercial enzyme-linked immunosorbent assay (ELISA), was successfully applied for the rapid quantification of specific antibodies against Trypanosoma cruzi in fresh human sera showing an excellent agreement with ELISA.


Assuntos
Anticorpos Antiprotozoários/sangue , Doença de Chagas/diagnóstico , Doença de Chagas/veterinária , Ensaio de Imunoadsorção Enzimática/métodos , Trypanosoma cruzi/isolamento & purificação , Animais , Doença de Chagas/sangue , Doença de Chagas/parasitologia , Cisteína Endopeptidases/análise , Cisteína Endopeptidases/genética , Cisteína Endopeptidases/imunologia , Doenças do Cão/sangue , Doenças do Cão/diagnóstico , Doenças do Cão/parasitologia , Cães , Glicoproteínas/análise , Glicoproteínas/genética , Glicoproteínas/imunologia , Humanos , Neuraminidase/análise , Neuraminidase/genética , Neuraminidase/imunologia , Proteínas de Protozoários/análise , Proteínas de Protozoários/genética , Proteínas de Protozoários/imunologia , Trypanosoma cruzi/genética , Trypanosoma cruzi/imunologia , Fatores de Virulência/sangue , Fatores de Virulência/genética , Fatores de Virulência/imunologia
19.
Mol Immunol ; 114: 497-512, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31518854

RESUMO

Compounding with the problem of frequent antigenic shift and occasional drift of the segmented genome of Avian Influenza Virus (AIV), vaccines based on major surface glycoproteins such as haemagglutinin (HA) to counter heterosubtypic AIV infection in chickens remain unsuccessful. In contrast, neuraminidase (NA), the second most abundant surface glycoprotein present in viral capsid is less mutable and, in some instances, successful in eliciting inter-species cross-reactive antibody responses. However, without selective activation of B-cells and T-cells, the ability of NA to induce strong cell mediated immune responses is limited, thus NA based vaccines cannot singularly address the risk of virus escape from host defence. To this end, the highly conserved ectodomain of influenza matrix protein-2 (M2e) has emerged as an attractive cross-protective vaccine target. The present study describes the potential of recombinant Lactococcus lactis (rL. lactis) in expressing functional influenza NA or M2e proteins and conferring effective mucosal and systemic immune responses in the intestine as well as in the upper respiratory airways (trachea) of chickens. In addition, lavages collected from trachea and intestine of birds administered with rL. lactis expressing influenza NA or M2e protein were found to protect MDCK cells against avian influenza type A/PR/8/34 (H1N1) virus challenge. Although minor, the differences in the expression of pro-inflammatory cytokines gene transcripts targeted in this study among the birds administered with either empty or rL. lactis could be attributed to the activation of innate response by L. lactis.


Assuntos
Galinhas/imunologia , Imunidade nas Mucosas/imunologia , Influenza Aviária/imunologia , Lactococcus lactis/imunologia , Neuraminidase/imunologia , Proteínas da Matriz Viral/imunologia , Animais , Anticorpos Antivirais/imunologia , Linfócitos B/imunologia , Galinhas/virologia , Reações Cruzadas/imunologia , Vírus da Influenza A Subtipo H1N1/imunologia , Vacinas contra Influenza/imunologia , Linfócitos T/imunologia , Vacinação/métodos
20.
Influenza Other Respir Viruses ; 13(5): 504-516, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-31411006

RESUMO

BACKGROUND: Formulation of neuraminidase (NA) within influenza vaccines is gaining importance in light of recent human studies. The enzyme-linked lectin assay (ELLA) is considered a reliable assay to evaluate human anti-NA antibodies. OBJECTIVES: To overcome interference by hemagglutinin (HA)-specific antibodies and detect neuraminidase inhibitory (NI) antibodies only, two different sources of antigen have been studied in ELLA: reassortant viruses with a mismatched avian origin-HA or Triton X-100 (Tx)-treated wild-type viruses. Pseudotypes or pseudovirus (PV), characterized by a lentivirus core bearing human influenza NA and avian influenza HA, were investigated as an alternative source of antigen and compared to HA-mismatched and Tx-treated viruses, since represent a safer product to be handled. METHODS: Two independent panels of sera were analyzed by ELLA to evaluate the anti-NA response against N1 (A/California/07/2009 (H1N1pdm)) and N2 (A/Hong Kong/4801/2014 (H3N2)). The NA inhibition (NI) antibody titers measured as either the 50% end point or 50% inhibitory concentration (IC50 ) were compared for every source of antigen. RESULTS: The ELLA assay performed well with all three sources of antigen. NI titers measured using each antigen type correlated well when reported either as end point titers or as the IC50 . CONCLUSIONS: This study suggests that HA-mismatched whole virus, Triton-treated wild-type virus or PV can be used to measure NI antibody titers of human sera, but further comparability/validation assays should be performed to assess statistical differences. The data support the use of PV as an attractive alternative source of antigen and justify further investigation to improve stability of this antigen source.


Assuntos
Antígenos Virais/imunologia , Ensaios Enzimáticos/normas , Vírus da Influenza A/imunologia , Lectinas/química , Neuraminidase/imunologia , Octoxinol/farmacologia , Animais , Anticorpos Antivirais/sangue , Antígenos Virais/genética , Aves/virologia , Ensaios Enzimáticos/métodos , Glicoproteínas de Hemaglutininação de Vírus da Influenza/imunologia , Humanos , Vírus da Influenza A Subtipo H1N1/efeitos dos fármacos , Vírus da Influenza A Subtipo H1N1/imunologia , Vírus da Influenza A Subtipo H3N2/efeitos dos fármacos , Vírus da Influenza A Subtipo H3N2/imunologia , Vírus da Influenza A/efeitos dos fármacos , Vacinas contra Influenza/imunologia , Influenza Aviária/virologia , Influenza Humana/virologia , Lentivirus/genética , Lentivirus/imunologia , Neuraminidase/antagonistas & inibidores , Vírus Reordenados/genética , Vírus Reordenados/imunologia
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